The uncharacterized locus C15orf65, which we previously identified as part of a translocation in Hodgkins lymphoma, encodes a small, highly conserved, 15kDa protein of completely unknown function. We generated a monoclonal antibody against the C terminus of C15orf65 and demonstrated that the protein is expressed in a cell-cycle-dependent fashion, with levels peaking in the G1/S phase of the cell cycle. Overexpression of the C15orf65 protein resulted in increased cell cycling, whereas knockdown decreased cell cycling and ablated the ability of myeloid cells to form tumors upon xenotransplantation. In myeloid and kidney cell lines, C15orf65 localized to the nucleus and to the chromatin fraction in particular, raising the consideration that it may participate in a histon-binding complex. However, its mechanism of action remains unexplored. Analysis of public microarray datasets showed C15orf65 mRNA expression in many tissues, with particularly high expression in the breast, trabecular bone osteoblasts, and hematopoietic stem and progenitor cells. C15orf65 mRNA levels are significantly upregulated in the PML-RAR? subtype of acute myeloid leukemia (AML) and the RARS subtype of myelodysplastic syndrome (MDS), indicating a potential role in malignant as well as normal hematopoiesis. C15orf65 is highly conserved across the animal kingdom, especially within the vertebrate lineage, and has additional homologs outside of the animal kingdom. This degree of conservation indicates selective pressure for a conserved function, which we postulate is mediated by C15orf65's principal domain, DUF4490. The closest relative of DUF4490 for which both structure and function have been determined is the histone-binding Tudor domain of Sgf29. However, the structure and function of DUF4490 are as yet completely uncharacterized and thus represent a potentially informative target for structural determination. The combination of C15orf65's conservation and localization with a cell cycling phenotype and involvement in multiple hematopoietic malignancies suggests that C15orf65 may be a previously undescribed epigenetic regulator with a particular role in governing cell cycling in hematopoietic cells. We propose to 1) Identify binding partners for C15orf65 to characterize its pathway interactions; 2) Determine crystal and NMR structures for C15orf65; and 3) Delineate the functional role of C15orf65 in hematopoiesis using a newly generated conditional knockout model for the murine homolog of C15orf65, Gm5918. Our investigation of C15orf65 will provide insight into the biophysical, biochemical, and functional properties of this novel gene, and may also lead us to a new pathway for cell cycle regulation and identification of a novel histone binding domain.